IMMUNOLOGICAL EXTRACT AND METHOD OF PRODUCTION

Abstract

The present invention relates to a method of preparing an extract from edible bird's nest, a bird's nest extract and uses of the bird's nest extract. The method comprises preparing an edible bird's nest (EBN) mixture; and contacting the mixture with an extraction solution to bind a molecule in the mixture, wherein the extraction solution comprises at least one binding moiety selected from the group comprising an opsonin binding moiety, a complement protein binding moiety, a lectin binding moiety, a ficolin binding moiety, a collectin binding moiety, and a pentraxin binding moiety.

Claims

1. A method of preparing an extract from edible bird's nest, the method comprising: (a) preparing an edible bird's nest (EBN) mixture; and (b) contacting the mixture with an extraction solution to bind a molecule in the mixture, wherein the extraction solution comprises at least one binding moiety selected from the group comprising an opsonin binding moiety, a complement protein binding moiety, a lectin binding moiety, a ficolin binding moiety, a collectin binding moiety, and a pentraxin binding moiety.

2. The method according to claim 1, wherein the mixture further comprises seaweed and/or hasma.

3. The method according to any one of claims 1 to 2, wherein the opsonin binding moiety has a molecular weight of 10 kDa to 750 kDa.

4. The method according to any one of claims 1 to 3, wherein the opsonin binding moiety comprises SEQ ID No. 1.

5. The method according to any one of claims 1 to 4, wherein the complement protein binding moiety has a molecular weight of 20 kDa to 7000 kDa.

6. The method according to any one of claims 1 to 5, wherein the complement protein binding moiety comprises SEQ ID No. 2.

7. The method according to any one of claims 1 to 6, wherein the lectin binding moiety has a molecular weight of 20 kDa to 1000 kDa.

8. The method according to any one of claims 1 to 7, wherein the lectin binding moiety comprises SEQ ID No. 3.

9. The method according to any one of claims 1 to 8, wherein the ficolin binding moiety has a molecular weight of 15 kDa to 900 kDa.

10. The method according to any one of claims 1 to 9, wherein the ficolin binding moiety comprises SEQ ID No. 4.

11. The method according to any one of claims 1 to 10, wherein the collectin binding moiety has a molecular weight of 15 kDa to 900 kDa.

12. The method according to any one of claims 1 to 11, wherein the collecting binding moiety comprises SEQ ID No. 5.

13. The method according to any one of claims 1 to 12, wherein the pentraxin binding moiety has a molecular weight of 20 kDa to 1000 kDa.

14. The method according to any one of claims 1 to 13, wherein the pentraxin binding moiety comprises SEQ ID No. 6.

15. The method according to any one of claims 1 to 14, wherein the at least one binding moiety comprises any one selected from: i) the lectin binding moiety and the opsonin binding moiety; ii) the lectin binding moiety, the complement protein binding moiety, the opsonin binding moiety, the ficolin binding moiety, the collectin binding moiety, and the pentraxin binding moiety; iii) the lectin binding moiety, the complement protein binding moiety, and the opsonin binding moiety; iv) the opsonin binding moiety, and the pentraxin binding moiety; v) the ficolin binding moiety, the collectin binding moiety, and the pentraxin binding moiety; vi) the complement protein binding moiety, the ficolin binding moiety, and the collectin binding moiety; and vii) the lectin binding moiety, the opsonin binding moiety, and the collectin binding moiety.

16. The method according to claim 15, wherein the at least one binding moiety comprises any one selected from: i) 70% lectin of the binding moiety and 30% opsonin of the binding moiety; ii) 20% lectin of the binding moiety, 20% complement protein of the binding moiety, 20% opsonin of the binding moiety, 20% pentraxin of the binding moiety, 10% ficolin of the binding moiety, and 10% collectin of the binding moiety; iii) 50% complement protein of the binding moiety, 25% lectin of the binding moiety and 25% opsonin of the binding moiety; iv) 50% opsonin of the binding moiety and 50% pentraxin of the binding moiety; v) 40% ficolin of the binding moiety, 30% collectin of the binding moiety and 30% pentraxin of the binding moiety; vi) 10% ficolin of the binding moiety, 20% collectin of the binding moiety and 70% complement protein of the binding moiety; and vii) 70% collectin of the binding moiety, 20% opsonin of the binding moiety and 10% lectin of the binding moiety.

17. The method according to any one of claims 1 to 16, wherein preparing the EBN mixture comprises washing the mixture, and filtering the washed mixture.

18. The method according to claim 17, wherein the washing step comprises exposing the mixture to a first enzyme solution, and soaking the mixture and the first enzyme solution in water.

19. The method according to claim 18, wherein the first enzyme solution comprises a nitrite reductase.

20. The method according to any one of claims 17 to 19, wherein preparing the EBN mixture comprises dipping the EBN mixture in oil prior to the contacting step.

21. The method according to any one of claims 17 to 20, wherein preparing the EBN mixture further comprises sterilising the washed EBN mixture prior to the contacting step.

22. The method according to any one of claims 1 to 21, wherein the contacting step is carried out in the presence of an ascorbic acid and a gold nanoparticle.

23. The method according to any one of claims 1 to 22, wherein the contacting step is carried out at between 4° C. to 37° C. for at least 20 minutes.

24. The method according to any one of claims 1 to 23, further comprising hydrolysing the bounded molecules with an acidic solution.

25. The method according to any one of claims 1 to 24, further comprising separating the at least one binding moiety and bounded molecules from the mixture; releasing the bounded molecules; and obtaining the released molecules by dialysis.

26. The method according to claim 25, further comprising treating the dialysed molecules with a second enzyme solution comprising a vegetable protease and/or a fruit protease.

27. The method according to claim 26, with at least one of the following conditions: (a) a concentration of the second enzyme solution is from about 10 μg/ml to about 100 μg/ml; and (b) treating of the isolated molecule with the second enzyme solution is carried out at 45° C. for 60 minutes at pH 6.5 to 9.0, and denaturing the second enzyme solution.

28. The method according to any one of claims 1 to 27, further comprising drying the molecules.

29. A bird's nest extract obtainable by the method according to any one of claims 1 to 28.

30. A bird's nest extract comprising a plurality of molecules selected from at least two groups comprising: opsonins, complement proteins, lectins, ficolins, collectins, and pentraxins.

31. The bird's nest extract according to claim 30, wherein the plurality of molecules is any one selected from: i) lectins and opsonins; ii) lectins, complement proteins, opsonins, ficolins, collectins, and pentraxins; iii) lectins, complement proteins, and opsonins; iv) opsonins and pentraxins; v) ficolins, collectins, and pentraxins; vi) complement proteins, ficolins, and collectins; and vii) lectins, opsonins, and collectins.

32. The bird's nest extract according to claim 31, wherein the plurality of molecules is any one selected from: i) 70% of lectins, and 30% of opsonins; ii) 20% of lectins, 20% of complement proteins, 20% of opsonins, 10% of ficolins, 10% of collectins, and 20% of pentraxins; iii) 25% of lectins, 50% of complement proteins, and 25% of opsonin; iv) 50% of opsonins, and 50% of pentraxins; v) 40% of ficolins, 30% of collectins, and 30% of pentraxins; vi) 70% of complement proteins, 10% of ficolins, and 20% of collectins; and vii) 10% of lectins, 20% of opsonins, and 70% of collectins.

33. The bird's nest extract according to any one of claims 30 to 32, wherein the molecules are hydrolysed by treatment with an acid solution and/or an enzymatic solution.

34. The bird's nest extract according to any one of claims 29 to 33, further comprising maltodextrin.

35. A composition comprising the bird's nest extract according to any one of claims 29 to 34; and a pharmaceutically-acceptable carrier, excipient or diluent.

36. A nutraceutical for use in modulating an immune system of a subject, the nutraceutical comprising the bird's nest extract according to any one of claims 29 to 34.

37. The bird's nest extract according to any one of claims 29 to 34 for use in medicine.

38. The bird's nest extract according to claim 37 for use in inhibiting dengue virus replication, or for use in modulating an immune system of a subject, or for use in inducing interferon regulatory factor 3 phosphorylation.

39. The bird's nest extract according to claim 38, wherein modulating the immune system is by inducing production of inflammatory cytokines, or by induction of NF-κB pathway and/or MAPK pathway.

40. Use of the bird's nest extract according to any one of claims 29 to 34 in the manufacture of a medicament.

41. A method for inhibiting dengue virus replication, or modulating the immune system in a subject, or inducing interferon regulatory factor 3 phosphorylation, the method comprises administering the bird's nest extract according to any one of claims 29 to 34.

Description

DETAILED DESCRIPTION

[0099] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs.

[0100] The terms “about”, “approximately”, “substantially” must be read with reference to the context of the application as a whole, and have regard to the meaning a particular technical term qualified by such a word usually has in the field concerned. For example, it may be understood that a certain parameter, function, effect, or result can be performed or obtained within a certain tolerance, and the skilled person in the relevant technical field knows how to obtain the tolerance of such term.

[0101] In the following description, numerous specific details are set forth in order to provide a thorough understanding of various illustrative embodiments of the invention. It will be understood, however, to one skilled in the art, that embodiments of the invention may be practiced without some or all of these specific details. It is understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention. In the drawings, like reference numerals refer to same or similar functionalities or features throughout the several views.

[0102] The human immune system protects the body from foreign organisms, and comprise an innate immune system and an adaptive immune system. The innate immune system provides immediate defence against infection, while the adaptive immune system provides a long-lasting immunity against specific foreign organisms. Parkin and Cohen (Lancet 2001, 357, 1777-1789) provided an overview of the immune system and the main components in the human body.

[0103] Major functions of the innate immune system includes recruiting immune cells to infection sites through the production of chemical factors including cytokines and to promote removal of the foreign organisms; activation of the complement cascade; and activation of the adaptive immune system.

[0104] An opsonin is any molecule that enhances phagocytosis, and includes antibodies, complement proteins, and circulating proteins (for example pentraxins, collectins and ficolins). An opsonin typically marks an antigen for an immune response or mark dead cells for recycling. Most, if not all, cell membranes maintain a non-zero transmembrane potential and makes it difficult for two cells to come together. Opsonins generally work by binding to their target cells and enhance phagocytosis, i.e. the opsonin serve as a linker. The complement system is a part of the immune system that enhances the ability of antibodies and phagocytic cells to clear microbes and damaged cells from organisms. The complement system comprises a number of complement proteins which circulate in the blood as inactive precursors and are activated by biochemical pathways. Pentraxins, collectins, and ficolins are soluble innate immune pattern-recognition proteins which identify non-self or altered-self molecular patterns on the surfaces of dying cells and promotes the programmed cell death (e.g. apoptosis) and the clearance of dying cells and cellular material by macrophages and other phagocytic cells. As defined above, the term “opsonin” in the context of the invention excludes complement proteins, lectins, ficolins, collectins and pentraxins.

[0105] Lectins are carbohydrate-binding proteins and perform recognition on the cellular and molecular level. Within the animal lectins, C-type lectins are the most abundant and are grouped into three major families: selectins, collectins and endocytic lectins. Collectins are believed to be involved in the pattern recognition of respiratory viruses and pathogenic bacteria. Examples include the collagenous lectins such as mannose binding proteins (MBP), pulmonary surfactant SP-A and SP-D and conglutinin. MBP is an example of a protective collectin that is able to bind oligomannose residues of bacterial and fungal cell surface oligosaccharides. MBP is also able to active the classical and the alternative complement pathways. Another endogenous collectin is a mannose receptor and is expressed on macrophage and dendritic cell surfaces, and are able to recognise and bind bacteria. As defined above, the term “lectin” in the context of the invention excludes ficolins and collectins, i.e. lectins other than ficolins and collectins.

[0106] FIG. 1 shows the method to extract and isolate the bioactive molecules of interest from edible bird's nest (EBN): [0107] (a) cleaning EBN to remove contaminants; [0108] (b) grounding the cleaned EBN and sifting through a mesh; [0109] (c) placing the EBN powder in water to provide an EBN mixture; hasma and/or seaweed may optionally be cleaned and added into the EBN mixture as well; [0110] (d) sterilising the EBN or EBN mixture; [0111] (e) dipping the EBN or EBN mixture into oil; [0112] (f) treating the EBN mixture with an extraction solution comprising at least one binding moiety, the binding moiety being selected from the group comprising an opsonin binding moiety, a complement protein binding moiety, a lectin binding moiety, a ficolin binding moiety, a collectin binding moiety, and a pentraxin binding moiety; [0113] (g) hydrolysing the bounded molecules partially with an acidic solution; [0114] (h) separating the binding moiety and bioactive molecules from the EBN mixture; [0115] (i) releasing the bounded molecules by addition of peptides of large molecular weight; [0116] (j) obtaining the released bioactive molecules via dialysis; [0117] (k) treating the isolated fraction from dialysis with a second enzymatic solution to further break down the bioactive molecules; [0118] (l) denaturing and removing the enzymes of the second enzymatic solution; [0119] (m) drying the isolated fraction to obtain a solid product.

[0120] Certain steps described may be omitted, or performed in a different order. For example, steps (a)-(e), individually or in combination, may be considered as steps in preparing the EBN mixture for treating (or contacting) with the extraction solution. Hydrolysis of the target molecules is beneficial to maximise their biological effects. The time and temperature of the process may be varied to determine optimal parameters depending on the enzyme, binding moiety, and bioactive molecules being extracted.

[0121] The crude EBN (1 piece is approximately 10 to 50 g) is cleaned by soaking in water to remove nitrites, mites and other contaminants. The other possible contaminants that are removed may include heavy metals, bleach and other minute debris, including stains.

[0122] An effective method to remove the nitrites is to use a solution containing nitrite reductase enzymes from fruits, plants and soil. Additionally, the solution may contain another enzyme to inactivate any accompanying bacteria that produce the nitrite. To remove mites, a solution containing special fruit proteinases are used. Such examples include any such protease from papaya (papain), kiwifruit (actinidin), pineapple (bromelain), fig (ficin) etc. These proteases may be used in any suitable concentration that will allow for the inactivation of the bacteria.

[0123] The EBN mixture was treated sequentially with each enzymatic solution for at least 5 minutes from room temperature to 40° C. Nanobubbling of the resultant suspension of EBN in the enzymatic solution will cause the degraded cellular debris to float to the surface of the water where it can be easily removed. The enzymatic solution is subsequently removed from the solid EBN. The solid EBN can be further washed to remove any residual enzymes and contaminants. The cleaned EBN is dried to remove excess water, preferably at 70° C. for 12 h.

[0124] The cleaned EBN is grounded and sifted through a mesh. The size of the mesh should be sufficient to remove any large impurities left, preferably in a size of 200 to 700 μm. Most preferably the mesh size is 600 μm.

[0125] The EBN powder is placed in water, preferably distilled or deionised water, at 5° C. for 5 hours. A suitable concentration is 25 g of EBN in 1000 mL of water. The mixture may be further sterilised at 121° C. for 10 to 20 minutes if desired. Hasma and/or seaweed may also be cleaned as above and soaked in the mixture with the EBN. Alternatively, the hasma and/or seaweed may be prepared separately and added to the EBN mixture. Subsequently, the EBN mixture is dipped into oil to enhance the binding in the subsequent treatment with the binding moiety through enhanced interaction and affinity of the binding moiety and the bioactive molecules.

[0126] The EBN mixture is treated with an aqueous solution containing at least one binding moiety in a temperature range from 4 to 37° C. for at least 20 minutes. With a temperature of 25 to 37° C., 20 to 120 minutes suffice, but could be kept longer overnight at a lower temperature. With a temperature of 4° C., the mixture of antibody and EBN is kept for at least 9 hours. Generally, the lower the temperature the longer the time required for the binding moiety solution to completely bind to the targeted compounds. The binding moiety is selected from the group comprising an opsonin binding moiety, a complement protein binding moiety, a lectin binding moiety, a ficolin binding moiety, a collectin binding moiety, and a pentraxin binding moiety. In an embodiment, the contacting or mixing step of the EBN mixture and binding moiety is carried out in the presence of ascorbic acid and/or gold nanoparticles. The ascorbic acid provides anti-oxidant properties and prevents or minimises the degradation of the bioactive molecules, for example from reactive oxygen species. The gold nanoparticles provide a stable and non-reactive environment which enhances the binding of the target molecules and binding moiety. The at least one binding moiety present in the extraction solution will bind to the targeted molecule and allow the bounded molecule to be extracted out of the EBN mixture.

Examples of Binding Moieties

[0127] An example of an opsonin binding moiety protein is GP-340, a putative opsonin receptor for lung surfactant protein D. The protein has the following sequence (SEQ ID No. 1) with a molecular weight of 260.79 kDa:

TABLE-US-00001    1 mgistvilem cllwgqvlst ggwiprttdy aslipsevpl dqtvaegspf psestlesta    61 aegspisles tlestvaegs lipsestles tvaegsdsgl alrlvngdgr cqgrveilyr   121 gswgtvcdds wdtndanvvc rqlgcgwams apgnawfgqg sgpialddvr csghesylws   181 cphngwlshn cghgedagvi csaaqpqstl rpeswpvris ppvptegses slalrlvngg   241 drcrgrvevl yrgswgtvcd dywdtndanv vcrqlgcgwa msapgnaqfg qgsgpivldd   301 vrcsghesyl wscphngwlt hncghsedag vicsapqsrp tpspdtwpts hastagpess   361 lalrlvnggd rcqgrvevly rgswgtvcdd swdtsdanvv crqlgcgwat sapgnarfgq   421 gsgpivlddv rcsgyesylw scphngwlsh ncqhsedagv icsaahswst pspdtlptit   481 1pastvgses slalrlvngg drcqgrvevl yrgswgtvcd dswdtndanv vcrqlgcgwa   541 mlapgnarfg qgsgpivldd vrcsgnesyl wscphngwls hncghsedag vicsgpessl   601 alrlvnggdr cqgrvevlyr gswgtvcdds wdtndanvvc rqlgcgwams apgnarfgqg   661 sgpivlddvr csghesylws cpnngwlshn cghhedagvi csaaqsrstp rpdtlstitl   721 ppstvgsess ltlrlvngsd rcqgrvevly rgswgtvcdd swdtndanvv crqlgcgwam   781 sapgnarfgq gsgpivlddv rcsghesylw scphngwlsh ncghhedagv icsysqsrpt   841 pspdtwptsh astagsessl alrlvnggdr cqgrvevlyr gswgtvcdds wdtsdanvvc   901 rqlgcgwats apgnarfgqg sgpivlddvr csgyesylws cphngwlshn cqhsedagvi   961 csaahswstp spdtlptitl pastvgsess lalrlvnggd rcqgrvevly qgswgtvcdd  1021 swdtndanvv crqpgcgwam sapgnarfgq gsgpivlddv rcsghesypw scphngwlsh  1081 ncghsedagv icsasqsrpt pspdtwptsh astagsessl alrlvnggdr cqgrvevlyr  1141 gswgtvcddy wdtndanvvc rqlgcgwams apgnarfgqg sgpivlddvr csghesylws  1201 cphngwlshn cghhedagvi csasqsqptp spdtwptsha stagsessla lrlvnggdrc  1261 qgrvevlyrg swgtvcddyw dtndanvvcr qlgcgwatsa pgnarfgqgs gpivlddvrc  1321 sghesylwsc phngwlshnc ghhedagvic sasqsqptps pdtwptshas tagsesslal  1381 rlvnggdrcq grvevlyrgs wgtvcddywd tndanvvcrq lgcgwatsap gnarfgqgsg  1441 pivlddvrcs ghesylwscp hngwlshncg hhedagvics asqsqptpsp dtwptsrast  1501 agsestlalr lvnggdrcrg rvevlyqgsw gtvcddywdt ndanvvcrql gcgwamsapg  1561 naqfgqgsgp ivlddvrcsg hesylwscph ngwlshncgh hedagvicsa aqsqstprpd  1621 twlttnlpal tvgsesslal rlvnggdrcr grvevlyrgs wgtvcddswd tndanvvcrq  1681 lgcgwamsap gnarfgqgsg pivlddvrcs gnesylwscp hkgwlthncg hhedagvics  1741 atqinstttd wwhpttttta rpssncggfl fyasgtfssp sypayypnna kcvweievns  1801 gyrinlgfsn lkleahhncs fdyveifdgs lnsslllgki cndtrqifts synrmtihfr  1861 sdisfqntgf lawynsfpsd atlrlvnlns syglcagrve iyhggtwgtv cddswtiqea  1921 evvcrqlgcg raysalgnay fgsgsgpitl ddvecsgtes tlwqcrnrgw fshncnhred  1981 agvicsgnhl stpapflnit rpntdyscgg flsqpsgdfs spfypgnypn nakcvwdiev  2041 qnnyrvtvif rdvqleggcn ydyievfdgp yrsspliary cdgargsfts ssnfmsirfi  2101 sdhsitrrgf raeyysspsn dstnllclpn hmqasysrsy lqslgfsasd lvistwngyy  2161 ecrpqitpnl viftipysgc gtfkqadndt idysnfltaa vsggiikrrt dlrihvscrm  2221 lqntwvdtmy iandtihvan ntiqveevqy gnfdvnisfy tsssflypvt srpyyvdlnq  2281 dlyvqaeilh sdavltlfvd tcvaspysnd ftsltydlir sgcvrddtyg pysspslria  2341 rfrfrafhfl nrfpsvylrc kmvvcraydp ssrcyrgcvl rskrdvgsyq ekvdvvlgpi  2401 qlqtpprree epr 

[0128] An example of a complement protein binding moiety protein is the complement receptor type 2 isoform 1 precursor [Homo sapiens]. The protein has the following sequence (SEQ ID No. 2) with a molecular weight of 119.18 kDa:

TABLE-US-00002    1 mgaagllgvf lalvapgvlg iscgspppil ngrisyystp iavgtvirys csgtfrlige    61 ksllcitkdk vdgtwdkpap kceyfnkyss cpepivpggy kirgstpyrh gdsvtfackt   121 nfsmngnksv wcqannmwgp trlptcvsvf plecpalpmi hnghhtsenv gsiapglsvt   181 yscesgyllv gekiinclss gkwsavpptc eearckslgr fpngkvkepp ilrvgvtanf   241 fcdegyrlqg ppssrcviag qgvawtkmpv ceeifcpspp pilngrhign slanvsygsi   301 vtytcdpdpe egvnfilige stlrctvdsq ktgtwsgpap rcelstsavq cphpqilrgr   361 mvsgqkdryt yndtvifacm fgftlkgskq ircnaqgtwe psapvcekec qappnilngq   421 kedrhmvrfd pgtsikyscn pgyvlvgees iqctsegvwt ppvpqckvaa ceatgrqllt   481 kpqhqfvrpd vnsscgegyk lsgsvyqecq gtipwfmeir lckeitcppp pviyngahtg   541 ssledfpygt tvtytcnpgp ergvefslig estirctsnd qergtwsgpa plcklsllav   601 qcshvhiang ykisgkeapy fyndtvtfkc ysgftlkgss qirckadntw dpeipvcekg   661 cqsppglhhg rhtggntvff vsgmtvdytc dpgyllvgnk sihcmpsgnw spsaprceet   721 cqhvrqslqe lpagsrvelv ntscqdgyql tghayqmcqd aengiwfkki plckvihchp   781 ppvivngkht gmmaenflyg nevsyecdqg fyllgekklq crsdskghgs wsgpspqclr   841 sppvtrcpnp evkhgyklnk thsayshndi vyvdcnpgfi mngsrvirch tdntwvpgvp   901 tcikkafigc ppppktpngn htggniarfs pgmsilyscd qgyllvgeal llcthegtws   961 qpaphckevn csspadmdgi qkgleprkmy qygavvtlec edgymlegsp qsqcqsdhqw  1021 npplavcrsr slapvlcgia aglilltfli vitlyviskh rarnyytdts qkeafhlear  1081 evysvdpynp as 

[0129] An example of a lectin binding moiety protein is the c Killer cell lectin-like receptor subfamily B member 1. The protein has the following sequence (SEQ ID No. 3) with a molecular weight of 25.42 kDa:

TABLE-US-00003   1 mdqqaiyael nlptdsgpes sspsslprdv cqgspwhqfa lklscagiil lvlvvtglsv   61 svtsliqkss iekcsvdiqq srnktterpg llncpiywqq lrekcllfsh tvnpwnnsla  121 dcstkessll lirdkdelih tqnlirdkai lfwiglnfsl seknwkwing sflnsndlei  181 rgdakensci sisqtsvyse ycsteirwic qkeltpvrnk vypds 

[0130] An example of a ficolin binding moiety protein has the following sequence (SEQ ID No. 4) with a molecular weight of 35.08 kDa:

TABLE-US-00004   1 melsgatmar glavllvlfl hiknlpaqaa dtcpevkvvg legsdkltil rgcpglpgap   61 gpkgeagvig ergerglpga pgkagpvgpk gdrgekgmrg ekgdagqsqs catgprnckd  121 lldrgyflsg whtiylpdcr pltvlcdmdt dgggwtvfqr rmdgsvdfyr dwaaykqgfg  181 sqlgefwlgn dnihaltaqg sselrvdlvd fegnhqfaky ksfkvadeae kyklvlgafv  241 ggsagnsltg hnnnffstkd qdndvsssnc aekfqgawwy adchasnlng lylmgphesy  301 anginwsaak gykysykvse mkvrpa 

[0131] An example of a collectin binding moiety protein is Collectin-12. The protein has the following sequence (SEQ ID No. 5) with a molecular weight of 81.53 kDa:

TABLE-US-00005   1 mkddfaeeee vqsfgykrfg iqegtqctkc knnwalkfsi illyilcall titvailgyk   61 vvekmdnvtg gmetsrqtyd dkltavesdl kklgdqtgkk aistnselst frsdildlrq  121 qlreitekts knkdtleklq asgdalvdrq sqlketlenn sflittvnkt lqayngyvtn  181 lqqdtsvlqg nlqnqmyshn vvimnlnnln ltqvqqrnli tnlqrsvddt sqaiqriknd  241 fqnlqqvflq akkdtdwlke kvqslqtlaa nnsalakann dtledmnsql nsftgqmeni  301 ttisqaneqn lkdlqdlhkd aenrtaikfn qleerfqlfe tdivniisni sytahhlrtl  361 tsnlnevrtt ctdtltkhtd dltslnntla nirldsyslr mqqdlmrsrl dtevanlsvi  421 meemklvdsk hgqliknfti lqgppgprgp rgdrgsqgpp gptgnkgqkg ekgepgppgp  481 agergpigpa gppgerggkg skgsqgpkgs rgspgkpgpq gssgdpgppg ppgkeglpgp  541 qgppgfqglq gtvgepgvpg prglpglpgv pgmpgpkgpp gppgpsgavv plalqneptp  601 apedngcpph wknftdkcyy fsvekeifed aklfcedkss hlvfintree qqwikkqmvg  661 reshwigltd serenewkwl dgtspdyknw kagqpdnwgh ghgpgedcag liyagqwndf  721 qcedvnnfic ekdretvlss al 

[0132] An example of a pentraxin binding moiety protein is the neuronal pentraxin receptor (Homo sapiens). The protein has the following sequence (SEQ ID No. 6) with a molecular weight of 52.86 kDa:

TABLE-US-00006   1 mkflavllaa gmlaflgavi ciiasvplaa sparalpgga dnasvasgaa aspgpqrsls   61 alhgaggsag ppalpgapaa sahplppgpl fsrflctpla aacpsgaqqg daagaapger  121 eellllqsta eqlrqtalqq eariradqdt ireltgklgr cesglprglq gagprrdtma  181 dgpwdspali leledavral rdridrleqe lparvnlsaa papvsavptg lhskmdqleg  241 qllaqvlale kervalshss rrqrqeveke ldvlqgrvae lehgssaysp pdafkisipi  301 rnnymyarvr kalpelyaft acmwlrsrss gtgqgtpfsy svpgqaneiv lleaghepme  361 llindkvaql plslkdngwh hiciawttrd glwsayqdge lqgsgenlaa whpikphgil  421 ilgqeqdtlg grfdatqafv gdiaqfnlwd haltpaqvlg ianctapllg nvlpwedklv  481 eafggatkaa fdvckgraka 

[0133] Examples of Suitable Extraction Solutions that May be Used with the Above Binding Moieties Include:

[0134] Extraction solution 1: 50% to 90% of the lectin binding moiety and 10% to 50% of the opsonin binding moiety;

[0135] Extraction solution 2: 10% to 30% of the lectin binding moiety, 10% to 30% of the complement protein binding moiety, 10% to 30% of the opsonin binding moiety, 5% to 15% of the ficolin binding moiety, 5% to 15% of the collectin binding moiety, and 10% to 30% of the pentraxin binding moiety;

[0136] Extraction solution 3: 5% to 45% of the lectin binding moiety, 35% to 65% of the complement protein binding moiety, and 5% to 45% of the opsonin binding moiety;

[0137] Extraction solution 4: 10% to 90% of the opsonin binding moiety, and 10% to 90% of the pentraxin binding moiety;

[0138] Extraction solution 5: 30% to 50% of the ficolin binding moiety, 10% to 50% of the collectin binding moiety, and 10% to 50% of the pentraxin binding moiety;

[0139] Extraction solution 6: 55% to 85% of the complement protein binding moiety, 5% to 15% of the ficolin binding moiety, and 10% to 30% of the collectin binding moiety; and

[0140] Extraction solution 7: 5% to 15% of the lectin binding moiety, 10% to 30% of the opsonin binding moiety, and 55% to 85% of the collectin binding moiety.

[0141] Extraction solution 8: 70% of the lectin binding moiety and 30% of the opsonin binding moiety;

[0142] Extraction solution 9: 20% of the lectin binding moiety, 20% of the complement protein binding moiety, 20% of the opsonin binding moiety, 10% of the ficolin binding moiety, 10% of the collectin binding moiety, and 20% of the pentraxin binding moiety;

[0143] Extraction solution 10: 25% of the lectin binding moiety, 50% of the complement protein binding moiety, and 25% of the opsonin binding moiety;

[0144] Extraction solution 11: 50% of the opsonin binding moiety, and 50% of the pentraxin binding moiety;

[0145] Extraction solution 12: 40% of the ficolin binding moiety, 30% of the collectin binding moiety, and 30% of the pentraxin binding moiety;

[0146] Extraction solution 13: 70% of the complement protein binding moiety, 10% of the ficolin binding moiety, and 20% of the collectin binding moiety; and

[0147] Extraction solution 14: 10% of the lectin binding moiety, 20% of the opsonin binding moiety, and 70% of the collectin binding moiety.

[0148] The percentage provided for each binding moiety is the percentage weight of each binding moiety relative to the total weight of the binding moieties present. The binding moiety/moieties may be dissolved in any suitable solvent or used as a mixture. Examples of solvent includes water and buffer solutions.

[0149] After the contacting or mixing step, the mixture is homogenised with a homogeniser, treated with an acidic solution, and heated to 100° C. to cause partial hydrolysis of the target compounds. The acid is preferably a food acid, for example ascorbic acid, citric acid, malic acid, acetic acid, tartaric acid, fumaric acid, and lactic acid. The mixture is cooled to room temperature and neutralised to a pH of 7.

[0150] After hydrolysis, he at least one binding moiety and bounded molecules can be separated from the mixture by any of the commonly known methods. Some of these methods include physicochemical fractionation, class-specific affinity and antigen-specific affinity. Physicochemical fractionation includes differential precipitation, size-exclusion or solid-phase binding of immunoglobulins based on size, charge or other shared chemical characteristics of antibodies. Class-specific affinity includes solid-phase binding of particular antibody classes (e.g. IgG) by immobilised biological ligands that have specific affinity to immunoglobulins. Antigen-specific affinity includes using specific antigens to purify antibodies through their specific antigen-binding domains.

[0151] The bounded compounds are released from the at least one binding moiety by adding excess larger peptides. For example, the larger peptides should have a minimum molecular weight of 50 kDa, and include natural glycoaminoglycans and other proteins.

[0152] The released compounds are subsequently isolated from the added peptides, enzymes and at least one binding moiety via the use of a dialysis bag.

[0153] Alternatively, the EBN containing mixture may be sequentially treated with extraction solutions comprising a different binding moiety, and separated to extract out the desired compounds sequentially. This ensures optimal use of the EBN, seaweed and/or hasma, and avoids wastage.

[0154] The isolated/concentrated compounds can be further hydrolysed with vegetable and/or food proteases at 45° C. for 1 hour at a pH of 6.5 to 9.0. The concentration of enzymes used should be at least 10 μg/mL for effective hydrolysis, and preferably up to 100 μg/mL. Examples of suitable enzymes include corn and maize terminal proteases. The enzymes are subsequently denatured by heating the mixture at 70° C. for 5 minutes. The enzymes precipitate out at a temperature above 55° C., hence the mixture can be filtered at a temperature above 55° C. to afford the desired compounds as a solution in the filtrate.

[0155] The solution of desired compounds is dried to give the compounds as a powder. Preferably, the compounds are dried by freeze drying or spray drying. The freeze drying is carried out by cooling the solution to a temperature between −180° C. to −70° C. with liquid nitrogen or dry ice, and submitting the frozen mixture to vacuum to sublime the ice. The freeze drying can be repeated if required to give a dried powdered product.

[0156] A bird's nest concentrate or extract obtained from the process described above comprise the bioactive molecules extracted from the process described. In some embodiments, the bioactive molecules may have been broken down and may be difficult to characterise. In some embodiments, the bioactive molecules may be largely intact.

[0157] As such, the bird's nest extract comprises a plurality of molecules selected from at least two of the following groups: opsonin, a complement protein, a lectin, a ficolin, a collectin, and a pentraxin. The bird's nest extract may further include extracts from hasma and/or seaweed.

[0158] In an embodiment, the bird's nest extract comprises any one selected from:

i) the lectin and the opsonin;
ii) the lectin, the complement protein, the opsonin, the ficolin, the collectin, and the pentraxin;
iii) the lectin, the complement protein, and the opsonin;
iv) the opsonin and the pentraxin;
v) the ficolin, the collectin, and the pentraxin;
vi) the complement protein, the ficolin, and the collectin; and
vii) the lectin, the opsonin, and the collectin.

[0159] In particular, the bird's nest extract comprises any one selected from: [0160] i) 50% to 90% of the lectin, and 10% to 50% of the opsonin [0161] ii) 10% to 30% of the lectin, 10% to 30% of the complement protein, 10% to 30% of the opsonin, 5% to 15% of the ficolin, 5% to 15% of the collectin, and 10% to 30% of the pentraxin; [0162] iii) 5% to 45% of the lectin, 35% to 65% of the complement protein, and 5% to 45% of the opsonin; [0163] iv) 10% to 90% of the opsonin, and 10% to 90% of the pentraxin; [0164] v) 30% to 50% of the ficolin, 10% to 50% of the collectin, and 10% to 50% of the pentraxin; [0165] vi) 55% to 85% of the complement protein, 5% to 15% of the ficolin, and 10% to 30% of the collectin; [0166] viii) 5% to 15% of the lectin, 10% to 30% of the opsonin, and 55% to 85% of the collectin. The percentage given is the percentage weight of the particular bioactive molecule relative to the total weight of bioactive molecules present.

[0167] The following bird's nest extracts 1 to 7 were prepared based on the process described above using the extraction solutions 8 to 14 respectively, and are reflective of the properties possess by extracts with ranges encompassing these examples:

[0168] Extract 1 was prepared using EBN and comprises: 70% of lectins, and 30% of opsonins;

[0169] Extract 2 was prepared using EBN, hasma and seaweed, and comprises: 20% of lectins, 20% of complement proteins, 20% of opsonins, 10% of ficolins, 10% of collectins, and 20% of pentraxins;

[0170] Extract 3 was prepared using EBN and seaweed, and comprises: 25% of lectins, 50% of complement proteins, and 25% of the opsonin;

[0171] Extract 4 was prepared using EBN and seaweed and comprises: 50% of opsonins, and 50% of pentraxins;

[0172] Extract 5 was prepared using EBN and comprises: 40% of ficolins, 30% of collectins, and 30% of pentraxins;

[0173] Extract 6 was prepared using EBN and hasma, and comprises: 70% of complement proteins, 10% of ficolins, and 20% of collectins;

[0174] Extract 7 was prepared using EBN and hasma, and comprises: 10% of lectins, 20% of opsonins, and 70% of collectins.

[0175] The dried powdered product may be mixed with other additives to give a food or pharmaceutical product. Alternatively, the product may be dissolved in water along with other additives.

[0176] The dried product may be mixed with maltodextrin in various formulations as follows:

1. 75% of EBN/EBN mixture concentrate/extract product and 25% maltodextrin;
2. 50% of EBN/EBN mixture concentrate/extract product and 45% maltodextrin;
3. 50% of EBN/EBN mixture concentrate/extract product and 50% maltodextrin;
4. 30% of EBN/EBN mixture concentrate extract product and 70% maltodextrin.

[0177] It may be seen that the product comprises EBN concentrate/extract product and maltodextrin. Preferably, 30 to 75 wt. % of the EBN concentrate/extract and 25 to 70 wt. % of maltodextrin.

[0178] Bioavailability of these bioactive molecules are generally very poor due to their high water solubility. Common administration routes via topical application to the skin and joints, or oral administration is hampered by the poor permeability through the skin or hydrophobic membranes in the intestine. It is difficult for the bioactive molecules to reach the requisite sites in the body to have the desired effect. Other methods of administration are available but is generally not suitable to be administered without a health professional. The hydrolysis of the bioactive molecules partially by acidic and/or enzymatic hydrolysis may prove to be beneficial to break down the molecules into more easily absorbable compounds making the extract more beneficial.

[0179] Especially with a suitable formulation, the respective proprietary immunological concentrates can be delivered in effective and useful doses to the sites of pain and inflammation.

[0180] Formulated in lipid forms, the present concentrate/extract may be used to produce a first economical product in the market that is transdermal and is composed of safe and sustainable bioactive molecules, purified/extracted from natural but abundant supplies of EBN/EBN mixture recycled crumbs.

[0181] Extracts 1 to 7 (E1-E7) were tested on macrophages/B cells cell culture in vitro to determine their effects on the production of Type-1 IFN, cytokine production, and dengue virus replication. Macrophages/B cells in this application refer to differentiated primary macrophages/B cells derived from bone-marrow progenitor's stem cells from mouse femurs. All these features are commonly used indicators of protective immune response of macrophages/B cells in vitro.

Methods and Results of Biological Assays

[0182] 1. Method of Cell Culture and Dengue Virus Replication Assays

Bone Marrrow-Derived Macrophage Culture

[0183] Bone marrow cells were obtained by injecting culture media into the femur and tibia. All cells were spun down by centrifuging 1000 rpm for 5 mins at 4° C. To eliminate erythrocytes, cells were treated with 1 ml of red blood cells lysis buffer for 5 mins by incubating on ice. The cells were washed in 10 ml culture media and collected by centrifuging at 1000 rpm for 5 mins at 4° C. Bone marrow cells were counted using haemocytometer and 106 cells were differentiated on 10 cm culture plate containing 10 ml of MCSF containing media for 6 days.

Dengue Virus Infection of Macrophages

[0184] Dengue type 1 virus (Singapore Strain S275/90, D1) was propagated in C6/36 cells. Bone marrow-derived macrophages (3×106) were seeded into each well of 6-well tissue-culture plate (NUNC). After overnight incubation, macrophages were infected with D1 at a multiplicity of infection (M01) of 1 for 2 days. For detection of negative-strand D1 RNA, total RNA was extracted from D1-infected cells using TRIzol (Invitrogen). 1 μg of total RNAs was subject to reverse transcription with primer 5′-GTGCTGCCTGTGGCTCCATC-3′, and was subsequently used as a template for synthesis of a PCR fragment with the primer pair 5′-AGAACCTGTTGATTCAACAGCACC-3′ and 5′-CATGGAAGCTGTACGCATGG-3′. For detection of GAPDH by Reverse-Transcriptase PCR, cellular cDNAs were synthesized from above total RNA with oligodT primer. GAPDH fragments were synthesized with the following primers, murine GAPDH, 5′-GACAACTTTGGCATTGTGGAA-3′ and 5′-CCAGGAAATGAGCTTGACA-3′, respectively.

[0185] Referring to FIG. 2, it may be seen that the middle lane with the macrophages treated with Extract 1 (E1) in the above assay shows the absence or weak presence of a white band, indicating that Extract 1 restricts the replication of the dengue virus in macrophages. This may be used as a means to control the dengue virus replication.

[0186] 2. Method of Cell Culture, Real-Time Genes Expression Quantitative-PCR (qPCR)

Bone Marrow-Derived Macrophage Culture

[0187] Bone marrow cells were obtained by injecting culture media into the femur and tibia. All cells were spun down by centrifuging 1000 rpm for 5 mins at 4° C. To eliminate erythrocytes, cells were treated with 1 ml of red blood cells lysis buffer for 5 mins by incubating on ice. The cells were washed in 10 ml culture media and collected by centrifuging at 1000 rpm for 5 mins at 4° C. Bone marrow cells were counted using haemocytometer and 106 cells were differentiated on 10 cm culture plate containing 10 ml of MCSF containing media for 6 days.

Quantitative Real-Time PCR

[0188] 2×106cells/m1 of bone marrow macrophages were seeded onto 6 well plate in Opti-MEM (Gibco, US). Cells were stimulated with 50 μg/ml Poly(I:C) (InvivoGen, US), 1 μg/ml LPS 0111:B4 (purified from LPS 0111:B4) and 1 μg/ml LPS 055:B5 (Sigma, US) for 2 hours before RNA extraction using TRIzol (Invitrogen, US). 1 μg cDNA was synthesized from total RNA with Superscript III First Strand Synthesis System (Invitrogen, US) according to manufacturer's protocol. qPCR was performed on Applied Biosystems QuantStudio 6 Flex Real Time PCR system with the cytokine specific primers.

[0189] Referring to FIG. 3, it can be seen that the positive controls elicit the production of IFNβ, TNFα, IL-10, IL-6, and IL-12 to different extents. Extract 2 (E2) elicit the production of IFNβ and inflammatory cytokines in macrophages, and provide a most complete immune balancing platform whereby key immune molecules could be harvested to influence the orchestration of various pro-inflammatory and anti-inflammatory molecules to achieve a balanced state of immune system, manifested not just by macrophages but other important cells as well. The symphony of immune molecules that would produce enhanced effects of prevention of microbial infections as well as better management of cancers. Extract 2 could possibly possess anti-dengue properties as well.

[0190] 3. Phosphorylation of IRF3 in Macrophages

[0191] Extract 3 (E3) when applied to macrophages induce phosphorylation of IRF3 in macrophages as shown in FIG. 4 panel B. The lane marked E3 in the western bolt analysis shows the phosphorylation of IRF3 same as the positive controls poly(I:C) and LPS. Panel A of FIG. 4 shows the negative control in the SF marked lane. Activated IRF3 has been linked with enhanced anti-cancer and viral prevention properties through interaction with IRF7. Modulated properly via the adoption of the different ranges of triggering immune peptides, this might also be employed successfully in post-myocardial infarction cardioprotection.

[0192] The negative control (SF) is the edible bird's nest which had not undergo the extraction process, and may be prepared by soaking the edible bird's nest in water and made up to the same mass concentration as the extract being tested. The SF control may be prepared similarly for the other assays.

[0193] 4. Induction of NF-κB and MAPK Pathway in Immune Cells

[0194] Extract 4 (E4) when applied to the immune cells elicit the induction of the NFκB and MAPK pathway in immune cells as shown in FIG. 5. It can be seen in the Western bolt analysis that the immune cells treated with Extract 4 induces phosphorylation of p65, JNK, ERK, and p38 as shown in the lanes marked with Extract 4 while those with the negative control (SF lane) do not show the phosphorylated band.

[0195] NF-κB plays a key role in regulating the immune response to infection. Incorrect regulation of NF-κB has been linked to cancer, inflammatory and autoimmune diseases, septic shock, viral infection, and improper immune development. NF-κB has also been implicated in processes of synaptic plasticity and memory.

[0196] MAPKs are involved in directing cellular responses to a diverse array of stimuli, such as mitogens, osmotic stress, heat shock and proinflammatory cytokines. They regulate cell functions including proliferation, gene expression, differentiation, mitosis, cell survival, and apoptosis.

[0197] Extract 4 may be used in enhanced management of the various forms of stress, heat shock, and septic shock.

[0198] 5. Induction of B Cells to Produce Cytokines

[0199] Extract 5 (E5) could induce B cells to produce cytokines in culture as shown in FIG. 6. It can be seen in FIG. 6 that the lane marked 10% Extract 5 (10% wt. of Extract 5 in aqueous solution) shows increased production of IFNβ, TNFα, IL-10, IL-6, and IL-12 compared to the negative control (media) and 10% SF (a negative control). Although pentraxin is associated with B cells, ficolins and collectins may not be. However, the presence of the three different type of bioactive molecules could induce a wide pool of different B cells to produce a huge diversity of cytokines to cope better with the very challenging various chronic diseases such as diabetes, arthritis, multiple sclerosis, etc.

[0200] 6. Dose-Dependent Effect on B Cells for Cytokine Production Extract 6 (E6) when applied to B cells shows dose-dependent effects on B cells for cytokine

[0201] production as shown in FIG. 7, in particular for IFNβ, IL-10, and IL-6. By replacing pentraxin with complement proteins, with a combination of immune peptides that may not be closely associated to B cells, dose dependent effects on B cells for cytokine production may be achieved. These dose dependent and prolonged overnight effects may be manifested using Extract 6 instead of Extract 5 above. The rationale is that since all ficolins, collectins and complement proteins are components of the non-humoral arm of innate immunity, the earlier stage of the innate immunity, their overall effects could be more sustainable as the span of operational period of this composition could be longer, since the time covered by the earlier stage could be more accounted for.

[0202] 7. Selective Induction of IFNβ, TNFα, and IL-6, but not 11-10 and IL-12, in B Cells

[0203] Extract 7 (E7) when applied to B cells selectively induce IFN β, TNF α, and IL-6 in B cells, but not 11-10 and IL-12, as shown in FIG. 8. The increased expression of IFNβ, TNFα, and IL-6 are marked with a box to indicate the beneficial effects of Extract 7.

[0204] The primary role of TNF is in the regulation of immune cells. TNF, being an endogenous pyrogen, is able to induce fever, apoptotic cell death, cachexia, inflammation and to inhibit tumorigenesis and viral replication and respond to sepsis via IL1 & IL6 producing cells.

[0205] Interferon beta (IFNβ) balances the expression of pro- and anti-inflammatory agents in the brain, and reduces the number of inflammatory cells that cross the blood brain barrier. Overall, therapy with interferon beta leads to a reduction of neuron inflammation. Moreover, it is also thought to increase the production of nerve growth factor and consequently improve neuronal survival.

[0206] Interleukin 6 (IL-6) is an interleukin that acts as both a pro-inflammatory cytokine and an anti-inflammatory myokine. Interleukin 6 is secreted by T cells and macrophages to stimulate immune response, e.g. during infection and after trauma, especially burns or other tissue damage leading to inflammation. IL-6 also plays a role in fighting infection. In addition, osteoblasts secrete IL-6 to stimulate osteoclast formation. Smooth muscle cells in the tunica media of many blood vessels also produce IL-6 as a pro-inflammatory cytokine. IL-6's role as an anti-inflammatory cytokine is mediated through its inhibitory effects on TNF-alpha and IL-1, and activation of IL-1ra and IL-10.

[0207] TNF, IFNβ and IL-6 together present a very novel but balanced immune modulating platform whereby there could be self-balancing of pro-inflammatory and anti-inflammatory factors. The novelty of combining the likely presence of opsonins, lectins and collectins (although they are associated peptides) in the immunological concentrate has yielded the ability of B cells to have induced expression of TNF, IFNβ and IL-6 together. When there is a balanced immune modulating platform, symptoms such as loss of weight, muscle atrophy, fatigue, weakness, significant loss of appetite, bone loss or even neurodegeneration would be alleviated. Cancer would also be less likely to occur as a result of a healthy and balanced immune system.

[0208] As can be seen from the assays and results discussed, the bird's nest Extracts 1 to 7 are able to induce various immune cells to increase production of cytokines, both pro and anti-inflammatory cytokines. This modulates the immune system as both types of cytokines are boosted balancing the cytokines present in the body. This prevents the overproduction of a particular cytokine.

[0209] The bird's nest extract whether as defined by the process or by the product itself, are suitable to be consumed as a nutraceutical or health supplement. It may potentially be usable as a medicament in certain compositions.

[0210] The process described herein allows for valuable bioactive molecules to be extracted from EBN, and provide for a cost effective and important source of immune boosters.

[0211] Whilst there has been described in the foregoing description preferred embodiments of the invention, it will be understood by those skilled in the field concerned that many variations or modifications in details of design or construction may be made without departing from the present invention.